Albumin is the most abundant plasma protein, generally constituting slightly over one-half of the total protein in mammalian plasma. In the human body, albumin has the important role of regulating the water balance between blood and tissues, and of functioning as a transport molecule for various compounds, such as bilirubin, fatty acids, cortisol, thyroxine and drugs like sulfonamides and barbiturates, that are only sparsely soluble in water. An albumin deficiency can restrict the transport of sparsely water soluble materials throughout the body and a deficiency is signaled in an individual by an abnormal accumulation of serous fluid, or edema. Therefore, it is clinically important to determine whether an individual has a deficiency of serum albumin.
Likewise, it is clinically important to determine if an individual is excreting an excess amount of protein. A normal functioning kidney forms urine in an essentially two step process. Blood flows through the glomerulus, or glomerular region of the kidney. The capillary walls of the glomerulus are highly permeable to water and low molecular weight components of the blood plasma. Albumin and other high molecular weight proteins cannot pass through these capillary walls and essentially are filtered out of the urine so that the protein is available for use by the body. The liquid containing the low molecular weight components passes into the tubules, or tubular region, of the kidney where reabsorption of some urine components, such as low molecular weight proteins; secretion of other urine components; and concentration of the urine occurs. AM a result, through the combined processes of the glomerulus and tubules, the concentration of proteins in urine should be minimal. Therefore, abnormally high amounts of albumin in urine must be detected and related to a physiological dysfunction.
A relatively high concentration of albumin in the urine of an individual usually is indicative of a diseased condition. For example, the average normal concentration of protein in urine varies from about 10 mg/dL (milligrams per deciliter) to about 20 mg/dL, with approximately one-fifth of the total urinary protein being serum albumin. However, in a majority of diseased states, urinary protein levels increase appreciably, such that albumin accounts for from about 60 percent to about 90 percent of the excreted protein. The presence of an abnormal increased amount of protein in the urine, known as proteinuria, is one of the most significant indicators of renal disease, and may be indicative of various other non-renal related diseases.
Therefore, in order to determine if an individual has an albumin deficiency or to determine if an individual excretes an excess amount of protein, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive protein detection assays have been developed. Furthermore, of the several different assay methods developed for the detection or measurement of protein in urine and serum, the methods based on dye binding techniques have proven especially useful because dye binding methods are readily automated and provide reproducible and accurate results.
In general, dye binding techniques utilize pH indicator dyes that are capable of interacting with a protein, such as albumin, and that are capable of changing color upon interaction with a protein absent any change in pH. When a pH indicator dye interacts with, or binds to, a protein, the apparent pK.sub.a (acid dissociation constant) of the indicator dye is altered and the dye undergoes a color transition, producing the so-called "protein-error" phenomenon. In methods utilizing the dye binding technique, an appropriate buffer maintains the pH indicator dye at a constant pH to prevent a color transition of the pH indicator dye due to a substantial shift in pH. Due to the "protein-error" phenomena, the pH indicator dye undergoes a color transition upon interaction with protein that is identical to the color change arising because of a change in the pH. Examples of pH indicator dyes used in the dry phase assay of proteins that are capable of interacting with or binding to proteins and exhibiting "protein-error" color transitions include tetrabromophenol blue (TBPB) and tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein.
Although pH indicator dyes have been used extensively in protein assays, several disadvantages still exist in protein assay methods utilizing indicator dyes. For example, methods based upon pH indicator dyes cannot sufficiently differentiate, quantitatively, between a trace protein concentration of about 15 to about 30 mg/dL and a negative protein concentration below about 15 mg/dL. A negative protein concentration is the normal background amount of protein present in urine, and is clinically insignificant. A trace protein concentration shows a slightly elevated amount of protein in urine and is clinically significant. An assay showing a trace amount of protein requires a confirmatory assay to conclusively show that an elevated amount of protein is present in the urine. Although several simple quantitative assays are available for the determination of the total protein content in a test sample, the majority of these assay methods, with the notable exception of the simple colorimetric reagent test strip, require the precipitation of protein to make quantitative protein determinations. Accordingly, the confirmatory assays are more time consuming and expensive than the test strip assays used to screen the urine samples for protein content. Therefore, a need exists for a test strip assay that substantially reduces the number of false positive assays for a trace amount of protein.
The colorimetric reagent test strip utilizes the previously discussed ability of proteins to interact with certain acid-base indicators and to alter the color of the indicator without any change in the pH. For example, when the indicator tetrabromophenol blue (TBPB) is buffered to maintain a constant pH of approximately 3, the test pad remains a yellow color upon contact with a test sample that does not contain protein. However, for test samples containing protein, the presence of protein causes the buffered dye to impart either a greenish-yellow color, a green color or a blue color to the test pad, depending upon the concentration of protein in the test sample. Consequently, the development of a greenish-yellow color in the test pad of a dry phase test strip can be interpreted as a trace amount of protein or as a negative amount of protein.
Some colorimetric test strips used in protein assays have a single test area consisting of a small square pad of a carrier matrix impregnated with a buffered pH indicator dye, such as tetrabromophenol blue. Other colorimetric test strips are multideterminant reagent strips that include one test area, or test pad, for the protein assay as described above, and further include several additional test pads on the same strip to permit the simultaneous assay of other urinary constituents, like pH. For both types of colorimetric test strips, the assay for protein in urine is performed simply by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test pad of the test strip to a standardized color chart provided on the colorimetric test strip bottle.
For test strips utilizing tetrabromophenol blue, buffered at pH 3, as the indicator dye, quantitative assays for protein can be performed and are reported as negative, trace, or one "plus" to four "plus". A negative reading, or yellow color, indicates that the urine contains less than about 15 mg/dL protein, as demonstrated by the lack of a color transition of the indicator dye. A trace reading, or greenish-yellow color, indicates that the urine contains from about 15 to about 30 mg/dL of protein. The one "plus" to four "plus" readings, signified by color transitions of green through increasingly dark shades of blue, are approximately equivalent to urine protein concentrations of 30, 100, 300, and over 2000 mg/dL protein, respectively, and serve as reliable indicators of increasingly severe proteinuria. Therefore, differentiating between a negative assay (yellow color) and a trace assay (greenish-yellow color) is important for an accurate protein analysis.
In accordance with the above-described method, an individual can readily determine, visually, that the protein content of a urine sample is in the range of 0 mg/dL to about 30 mg/dL. However, the color differentiation afforded by the presently available commercial test strips is insufficient to allow an accurate determination of urinary protein content between a sample having less than about 15 mg/dL protein (negative) and a sample including from about 15 to about 30 mg/dL protein (trace). The inability to differentiate between low urinary protein concentrations is important clinically because a healthy person usually has a urine protein level in the range of about 2 mg/dL to about 20 mg/dL. Therefore, it is clinically important to determine precisely the urine protein content of an individual, rather than merely estimating the protein content at some value less than about 30 mg/dL.
A trace reading for urinary protein is considered a positive assay, and confirmation of a positive test strip reading is required. The prevalent method of confirming a test strip positive protein assay is the turbidimetric sulfosalicylic acid method, abbreviated as SSA. A high frequency of false positive assays, requires confirmatory testing for each false positive assay, and the attendant added cost. Therefore, it is important that a screening test for protein, like a test strip assay, provide a low frequency of false positive readings.
Trace proteinuria is defined as protein excretion slightly above normal proteinuria. Normal excretion of protein is 50-150 mg/24 hours and 200-300 mg/24 hours in pregnancy. Using an average urine volume of 1250 mL/24 hrs, concentration units of 4-12 mg/dL (16-20 mg/dL in pregnancy) are calculated. Since 24 hour urine volumes vary from about 700 mL to more than 2000 mL, the range of normal protein is considerably wider, and, as expected, the more concentrated urine samples of higher specific gravity (SG) contain more protein. Trace proteinuria, then, is the concentration of protein that falls between negative and one "plus" (30 mg/dL). However, since normal proteinuria covers a range of protein concentrations, trace proteinuria also covers a range of protein concentrations. The trace protein concentration also is dependent on the specific gravity, or SG, of the sample.
The variation of protein concentration with SG does not effect the confirmatory SSA assay, and therefor protein precipitation in the SSA assay is considered indicative of clinical proteinuria. However, the SSA method also has limitations because it is a qualitative method and because procedures vary between clinical laboratories. Accordingly, the problem with a trace reading for protein provided by a screening test, such as a dry phase test strip, is that the reading must correspond to a protein range that is not well defined for clinical samples; that is dependent on the SG of the sample; and that overlaps with the normal, or negative, protein range. Further, trace readings are usually confirmed as positive by a qualitative method that has not been standardized, but is the method of choice in the art because the method is easy, requires no instrumentation, and detects clinical proteinuria in the presence of normal protein.
Of course, the protein content of a urine sample can be determined more precisely by quantitative 24 hour protein precipitation techniques. However, these tests are time consuming and relatively expensive. Furthermore, the precipitation tests must be run in a laboratory by trained personnel, and therefore are unavailable for the patient to perform at home to quickly determine urine protein content and to monitor the success or failure of a particular medical treatment.
Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying urine for protein content that allows visual differentiation of protein levels in the ranges of 0 mg/dL to about 15 mg/dL and about 15 mg/dL to about 30 mg/dL, and upwards to between about 100 mg/dL to about 300 mg/dL. By providing such an accurate method of determining urine protein concentration in an easy to use form, such as a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate test results, such that a diagnosis can be made without having to wait up to one day for assay results and medical treatment can be commenced immediately. In addition, the test strip method can be performed by the patient at home to more precisely monitor low levels of protein in urine and monitor the success of the medical treatment the patient is undergoing, without providing a large number of false positive assays that require unnecessary, time consuming and costly confirmatory testing. Finally, the method and composition used in a protein assay should be independent of the specific gravity of the urine to provide an accurate protein assay.
For example, the current urinary protein reagent test strips contain an octahalosulfophthalein protein indicator, e.g., tetrabromophenol blue (TBPB), as the indicator dye. When these strips are dipped into albumin-free urine samples of low to medium specific gravity, e.g., SG less than 1,020, the strips turn to a yellowish-green color. When the same strips are dipped into an albumin-free, high SG urine sample, e.g., SG equal to or greater than 1,020, the strips turn to a greenish-yellow color. This greenish-yellow color easily can be interpreted incorrectly as a clinically significant trace concentration of albumin (10-15 mg/dL). However, even with low SG urine samples, the negative color is difficult to differentiate from a true trace color.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy protein assay of urine by utilizing a test strip that includes a test pad comprising a carrier matrix incorporating a new and improved indicator reagent composition. Surprisingly and unexpectedly, the indicator reagent composition of the present invention essentially eliminates the interfering effects of specific gravity on the assay of urine samples including negative to trace amounts of protein. The new and improved indicator reagent composition of the present invention enhances visual color resolution by essentially eliminating the development of an interfering greenish-yellow color by high SG test samples including a negative amount of albumin. Therefore the sensitivity of the assay is enhanced, allowing urine protein concentrations to be determined accurately at levels of about 30 mg/dL or less, and precluding costly confirmatory testing arising from a false positive screening assay for albumin. In addition, the method of the present invention also can be used to determine the presence or concentration of higher concentrations of proteins, such as from about 100 mg/dL to about 2000 mg/dL, in a test sample.
Proteinuria resulting from abnormally high albumin levels depends upon the precise nature of the clinical and pathological disorder and upon the severity of the specific disease. Proteinuria can be intermittent or continuous, with transient, intermittent proteinuria usually being caused by physiological or functional conditions rather than by renal disorders. Therefore, accurate assays of urine and other test samples for protein must be available for both laboratory and home use. The assays must permit the detection or measurement of the proteins of interest, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the protein assay method could be utilized in a dip-and-read format for the easy and economical, qualitative or quantitative determination of protein in urine or other test samples.
Furthermore, any method of assaying for protein in urine or other test samples must yield accurate, trustworthy and reproducible results by utilizing a method that provides a detectable or measurable color transition as a result of an interaction between the indicator reagent composition and the protein, and not as a result of a competing chemical or physical interaction, such as a pH change or preferential interaction with a test sample component other than protein. Moreover, it would be advantageous if the protein assay method is suitable for use in dry reagent strips for the rapid, economical and accurate determination of protein in urine and other test samples. Additionally, the method and test pad, comprising the carrier matrix and the indicator reagent composition, utilized in the assay for protein, and the new indicator reagent composition, should not adversely affect or interfere with the other test reagent pads that are present on multideterminant test pad strips.
Although a dry phase chemistry test strip utilizing a dye, such as tetrabromophenol blue or tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein, has been used extensively for several years, no dry phase test strip has utilized a test pad comprising a carrier matrix, such as a filter paper, homogeneously impregnated with an indicator reagent composition including a hydrophobic polymeric compound as depicted above in general structural formula (I). The indicator reagent composition responds to urinary proteins and is essentially independent of urine specific gravity, thereby essentially eliminating the development of an interfering greenish-yellow color in the test pad by high SG samples including a negative amount of protein. Therefore, the assay exhibits an improved visual color resolution and an increased assay sensitivity, especially at lower protein concentration levels, to substantially reduce the number of false position assays. Surprisingly and unexpectedly, because of the essential elimination of the interferences related to urine specific gravity, the method of the present invention facilitates the dry phase test strip assay of urine and other test sample for albumin, especially at albumin levels of 0 mg/dL to about 30 mg/dL.
The prior art contains numerous references relating to the wet phase and the dry phase chemistry utilized in the pH indicator dye method of assaying urine for proteins. For example, Keston U.S. Pat. No. 3,485,587 discloses the basic dye binding technique used to assay for proteins at a constant pH. Keston teaches utilizing a single indicator dye, maintained at a constant pH slightly below the pK.sub.a (acid dissociation constant) of the dye and impregnated into a dry test paper, like filter paper, to determine the presence or concentration of albumin by monitoring the color transition of the dye. Free et al., in U.S. Pat. No. 3,095,277, also disclose a method of detecting the albumin content of liquid test samples by incorporating a suitable indicator composition into a bibulous carrier, like untreated filter paper. Similarly, Atkinson et al., in U.S. Pat. No. 3,438,737, disclose a test device comprising a test composition impregnated into an untreated bibulous matrix, such as filter paper, wood strips, synthetic plastic fibrous materials, nonwoven fabrics and woven fabrics for detecting protein in fluids.
Rittersdorf et al., in U.S. Pat. No. 4,013,416, disclose a test strip wherein an absorbent carrier is impregnated with an octahalosulfophthalein pH indicator dye, a buffer and a water-insoluble polypropylene glycol having a molecular weight of from about 500 to about 10,000 daltons. Rittersdorf et al. teach that the water-insoluble polypropylene glycol reduces the reactivity of the indicator dye with nitrogen containing compounds, such as metabolites of pharmaceuticals, thereby reducing the blank reaction in test strips. Rittersdorf et al. also teach only that water-insoluble propylene glycols are useful, e.g. polyethylene glycols are not useful. Rittersdorf et al. do not teach or suggest the usefulness of a polymer including a hydrocarbon, or essentially a hydrocarbon, backbone including pendant polyoxyalkylene units. In contrast, the indicator reagent composition of the present invention includes a hydrophobic polymeric compound having a hydrocarbon, or essentially hydrocarbon, backbone including 1 to about 8 alkylphenol units, like nonylphenol, linked by a methylene group or oxygen group, wherein the phenol moiety of each alkylphenol unit is ethoxylated and/or propoxylated to include about 2 and up to about 20 ethoxy and/or propoxy units in total.
The above-cited references do not teach or suggest, either alone or in combination, that an indicator reagent composition including a hydrophobic polymeric compound, as depicted above in general structural formula (I), can be used in a diagnostic device to achieve a more accurate determination of the amount of an analyte, like protein, and especially low amounts of an analyte, in a test sample. The references also do not teach or suggest, alone or in combination, that such an indicator reagent composition substantially reduces the number of false positive assays for albumin by essentially eliminating the effects of urine specific gravity in the assay for urinary proteins.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the method of the present invention provides increased sensitivity in the detection and measurement of proteins in a liquid test sample, such as a biological fluid, like urine. Surprisingly and unexpectedly, by utilizing an indicator reagent composition, comprising an indicator dye, a buffer and a hydrophobic polymeric compound depicted by general structural formula (I), homogeneously impregnated into a carrier matrix, an assay of a test sample including a negative amount of protein (e.g., less than about 15 mg/dL) can be differentiated from an assay of a test sample including a trace amount of protein (e.g., about 15 to about 30 mg/dL) more accurately. Accordingly, the number of false positive assays is reduced substantially, and the number of unnecessary and costly confirmatory assays also is reduced. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase test strip assay of urine and other test samples for proteins by utilizing a test pad, comprising a carrier matrix having homogeneously incorporated therein an indicator reagent composition comprising an indicator dye, a buffer and a hydrophobic polymeric compound of general structural formula (I), that provides an accurate protein assay for samples including a negative to low trace amount of protein, and that is independent of the specific gravity of the test sample.